Abstract
Abstract
Centromeres are specialized chromosomal regions essential for faithful chromosome segregation and genome stability. Their highly repetitive DNA composition and epigenetically defined chromatin state present significant challenges for genome‐wide profiling approaches such as chromatin immunoprecipitation followed by sequencing (ChIP‐seq). Standard ChIP‐seq workflows often perform poorly at centromeric regions, resulting in low signal‐to‐noise ratios and limited reproducibility. This article describes a ChIP‐seq protocol optimized to enrich for and analyze centromeric chromatin from cell lines. The procedure builds on established ChIP‐seq methodologies while incorporating centromere‐focused modifications in nuclei isolation, chromatin fragmentation, immunoprecipitation, library preparation, and data analysis. Detailed step‐by‐step instructions, critical parameters, recommended controls, and troubleshooting guidance are provided to support reproducible implementation. When applied carefully, this protocol enables reliable profiling of centromere‐associated chromatin and can be integrated into broader studies of chromosome organization, centromere function, and genome stability. © 2026 The Author(s).
Current Protocols
published by Wiley Periodicals LLC.
Basic Protocol 1
: Sucrose gradient–based isolation of nuclei
Support Protocol 1
: Titration of micrococcal nuclease (MNase)
Basic Protocol 2
: MNase digestion and immunoprecipitation of CENP‐A chromatin
Alternate Protocol 1
: Chromatin sonication and immunoprecipitation of CENP‐C chromatin complexes
Support Protocol 2
: NET blocking and washing of Protein G Sepharose beads
Basic Protocol 3
: DNA extraction
Alternate Protocol 2
: Reversal of cross‐links and DNA purification
Support Protocol 3
: Bead clean‐up
Basic Protocol 4
: Library preparation and size selection
Support Protocol 4
: qPCR to assess α‐satellite DNA enrichment
Basic Protocol 5
: ChIP‐seq data processing and analysis